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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1857-1865.)
© 1995 American Heart Association, Inc.

Articles

In Vivo Activation of met Tyrosine Kinase by Heterodimeric Hepatocyte Growth Factor Molecule Promotes Angiogenesis

Francesca Silvagno; Antonia Follenzi; Marco Arese; Maria Prat; Enrico Giraudo; Giovanni Gaudino; Giovanni Camussi; Paolo M. Comoglio; Federico Bussolino

From the Dipartimento di Genetica, Biologia e Chimica Medica (F.S., M.A., E.G., F.B.), the Institute for Cancer Research and Treatment (A.F., M.P., G.G., P.M.C.), and the Laboratorio di Immunopatologia (G.C.), Università di Torino, and the Cattedra di Nefrologia, II Facoltà di Medicina, Università di Pavia, Varese (G.C.), Italy.

Correspondence to Dr F. Bussolino, Dipartimento di Genetica, Biologia e Chimica Medica, Università di Torino, Via Santena 5bis, 10126 Torino, Italy.

	Abstract

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Abstract Hepatocyte growth factor (HGF) is a powerful motogen and mitogen for epithelial cells. The factor is a 90-kD heterodimer composed of an chain containing four kringle motifs and a ß chain showing structural homologies with serine proteases. It is, however, devoid of enzymatic activity. Recently, it has been reported that HGF activates migration and proliferation of endothelial cells and is angiogenic. In this article we discuss (1) the molecular domains of HGF required to activate in vitro and in vivo endothelial cells, studied by use of molecular mutants, and (2) the characteristics of the angiogenic response to HGF in an experimental model system of implanted reconstituted basement membrane (Matrigel). Two groups of mutants were made and used in vitro and in vivo: one with deletions of kringle domains and one with substitution at the cleavage site of the HGF precursor. In vitro, HGF variants containing only the first two (HGF-NK2) or the first three kringles (HGF-NK3) of the chain did not induce proliferation of endothelial cells even if used at a concentration 160-fold higher than that optimal for HGF (0.05 nmol/L). High concentrations of these mutants (4 to 8 nmol/L) activated a little endothelial cell motogenic response that was 60% lower than that elicited by HGF. Substitution of Arg 489 with Gln 489 in the HGF precursor generated an uncleavable single-chain factor, unable to induce either endothelial cell migration or proliferation. In vivo, HGF induced a dose-dependent angiogenic response, which was enhanced by heparin. Optimal HGF concentration (0.42 nmol/L) induced the appearance of clusters of migrating endothelial cells after 2 days. Canalized vessels appeared after 4 days, and the angiogenic response was completed within 6 days with full vascularization of the implanted Matrigel plug. HGF-NK2 and HGF-NK3 did not induce angiogenesis when used at equimolar, biologically active HGF concentrations. A little angiogenic response was observed at a concentration 10-fold higher than that of HGF. The uncleavable single-chain molecule was devoid of activity. The transcript of the HGF receptor was present in the Matrigel plug containing HGF, and the angiogenic response involved its activation, as shown by the agonist effect elicited by a monoclonal antibody against the extracellular domain of the receptor. Furthermore, [3-(1,4,-dihydroxytetralyl)-methylene-2-oxindole], a novel tyrosine kinase inhibitor effective on the HGF receptor, inhibited HGF-induced angiogenesis. During the formation of the new vessels, HGF induces expression of other angiogenic factors and chemokines: these include placental growth factor, vascular endothelial growth factor, KC, JE, macrophage inflammatory protein–2, and HGF itself. A neutralizing antibody to vascular endothelial growth factor partially prevented the angiogenesis induced by HGF. The results of this study demonstrate that the angiogenic response induced by HGF in vivo is elicited by stimulation of the HGF receptor, requires the presence of both and ß chains, and is amplified by other molecules, including vascular endothelial growth factor.

Key Words: growth factors • angiogenesis

	Introduction

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HGF is a heparin-binding glycoprotein produced by mesenchymal cells that triggers specific genetic programs of epithelial cells leading to organogenesis and tissue regeneration.^{1 2} HGF is a disulfide-linked heterodimer composed of a 55- to 65-kD subunit and a 32- to 36-kD subunit. The HGF gene encodes a single 92-kD protein^{3 4 5 6 7} containing a cleavage site after Arg 494.^{3 4 5 6 7} The and ß chains are originated by a proteolytic process catalyzed by the serine protease urokinase,⁸ a factor IX–like compound,^{9 10} or by thrombin.¹¹ The chain consists of an N-terminal hydrophobic leader sequence, a putative hairpin loop, and four kringle domains. The ß chain has homology with the catalytic domain of serine proteases but lacks enzymatic activity because of the loss of two essential amino acids in the catalytic site.⁴

HGF induces proliferation and motility of epithelial cells by stimulating the tyrosine kinase activity of its specific receptor encoded by the met proto-oncogene.⁷ The HGF receptor is a widely expressed 190-kD heterodimer composed of a 50-kD chain covalently linked to a 145-kD ß chain. The chain is extracellular, whereas the ß chain has an extracellular domain involved in HGF binding, a transmembrane domain, and a cytoplasmic tyrosine kinase domain.^{12 13 14 15} Ligand binding induces kinase activation and autophosphorylation of tyrosine residues located in the ß chain. These act as docking sites for recruitment of intracellular signal transducers containing SH2 domains.¹⁶ Cells transfected with met cDNA and expressing the functional receptor respond to HGF with a full spectrum of biological effects.^{17 18} HGF also binds to a low-affinity binding site contributed by the heparin sulfate proteoglycans of the cell membrane and of the pericellular matrix.^{7 19}

Recent data suggest that the spectrum of HGF activities is not restricted to epithelial cells but is extended to specific cell lineages of mesenchymal origin, including vascular ECs,^{19 20 21 22 23 24} and hemopoietic precursors.²⁵ ECs express the HGF receptor, which is phosphorylated on tyrosine residues of the ß chain upon HGF binding. HGF-induced receptor phosphorylation triggers migration and proliferation of ECs originated by different anatomic districts and animal species, including humans.¹⁹ Furthermore, HGF induces the in vitro organization of ECs into capillary-like structures^{19 26 27} and the expression of urokinase,²⁸ which is important for matrix degradation and EC invasion, and promotes the in vivo generation of new vessels.^{19 26}

Angiogenesis is a complex phenomenon that requires a series of biological events, namely matrix degradation, cell migration, cell proliferation, and differentiation, to form capillary structures.²⁹ All these steps are actively stimulated by HGF. The factor itself is a huge molecule composed of subunits and containing different structural motifs. To define the HGF domains required to elicit angiogenesis, we investigated the effect of the wild-type molecule and its mutants on EC proliferation and migration and in an angiogenesis assay in mice.

	Methods

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HGF and HGF Mutants
Full-length HGF cDNA was cloned from human liver mRNA⁸ and inserted as a BamHI-EcoRI fragment into the baculovirus transfer vector PVL1393 (Invitrogen). The recombinant vector was cotransfected with the Bsu I–digested BacPak6 viral DNA (Clontech) into Spodoptera frugiperda insect cells (Sf9) by use of the lipofectin procedure. Positive viral clones isolated by dot-blot hybridization and plaque assay were used for large-scale infection. HGF was obtained from the culture supernatant of Sf9-infected cells 72 hours after infection by affinity chromatography on a heparin–Sepharose FPLC column (BioRad Laboratories), eluted with a linear 0.5- to 1.8-mol/L NaCl gradient. The unprocessed recombinant factor (pro-HGF) was detected by Coomassie Blue staining as a 90-kD band in SDS polyacrylamide gel electrophoresis and cleaved by overnight incubation with 0.1% FCS.

HGF-NK2, the two-kringles variant of human HGF, was obtained by site-directed mutagenesis of HGF cDNA by use of the Altered Sites mutagenesis kit (Promega Biotech). The specific oligonucleotide 5'-ACATGCGCTGACTAATACTATGAA-3' inserted a thymidine in position 855 of the HGF cDNA sequence, yielding an Ochre (TAA) codon for arrest of translation. The specific oligonucleotide 5'-TGTTCCTTTGGTACCAACTGAATGC-3' generated in position 937 a Kpn I site suitable for further subcloning. The mutations induced in HGF cDNA were verified by DNA sequencing with the dideoxynucleotide method³⁰ by use of T7 DNA polymerase (Pharmacia Biotech). HGF-NK3, the three-kringle variant of human HGF, was a truncated form, encoded by a clone isolated during the cloning procedure.¹⁹ The uncleaved variant (Gln 489–HGF) was obtained by site-directed mutagenesis as previously reported.¹⁹ All the variant cDNAs were transiently expressed by use of the eukaryotic expression vector pBat in Neuro2A cells as previously described.¹⁹ Supernatants were collected 72 hours after transfection, tested for scatter activity on MDCK cells, and used for stimulation experiments. The scatter activity for HGF-NK2 and HGF-NK3 was 15 U/mL; 1 U was defined as the highest dilution that clearly dissociates MDCK cells and corresponded to 0.2 ng, 6 ng, and 6 ng of protein in standard preparations of HGF, HGF-NK2, and HGF-NK3, respectively.^{19 31}

Murine mAbs to Anti-HGF Receptor
The murine mAb DO-24 directed against the extracellular domain of the HGF receptor was obtained by immunization of mice with living cells of the human gastric carcinoma cell line GTL-16.^{32 33} As previously reported, it recognizes a C-terminal truncated soluble isoform of the HGF receptor, generated at the surface of living cells,³³ and reacts with fixed, impermeabilized cells.³⁴ Although produced against human cells, this mAb displays extensive cross-species specificity (Ref 34 and M. Prat et al unpublished data, 1995a). DO-24 does not compete with ¹²⁵I-HGF for receptor binding, and at nanomolar concentrations mimics HGF functions. In MDCK cells, it stimulates motility and receptor tyrosine kinase activity of the HGF receptor (M. Prat et al, unpublished data, 1995b). mAb DQ-13 was obtained by immunization of mice with a peptide corresponding to 19 C-terminal amino acids of the HGF receptor sequence. It reacts with the intracellular domain of the HGF receptor.³²

Cells
Human ECs from umbilical cord vein, prepared and characterized as previously described,¹⁹ were grown in M199 medium (GIBCO) supplemented with 20% FCS (Irvine), endothelial cell growth factor (100 g/mL) (Sigma Chemical Co), and porcine heparin (Sigma) (100 mg/mL). They were used at early passages (2 to 5). The mouse endothelioma cell line t.End.1 (kindly donated by Dr E.F. Wagner, Institute of Molecular Pathology, Wien, Austria), derived from a thymic hemangioma, expresses the polyoma middle T antigen, and has morphological and functional features of microvascular endothelial cells.³⁵ This line was maintained in DMEM (GIBCO) supplemented with 10% FCS and 750 mg/L G418 (GIBCO).

Cell Growth Assay
Human ECs (2.5x10³) were plated in 96-well plates (Costar) coated with 0.05% gelatin (Sigma) for 1 hour at 22°C in M199 medium containing 20% FCS. After 24 hours the medium was removed and replaced with M199 medium containing 5% FCS with or without factors. t.End.1 cells (1.2x10³) were plated in DMEM containing 10% FCS. After 24 hours the experiments were done in Iscove's medium (GIBCO) supplemented with 6 mg/L transferrin, 5 mg/L insulin, 100 mg/L soybean lecithin, 6.73 µg/L sodium selenite, and 400 mg/L BSA (Sigma; low content of endotoxin). Stimuli, indicated in "Results," were added every 2 days. Numbers of ECs and t.End.1 cells were estimated at days 6 and 8, respectively, by use of a colorimetric method as previously described.¹⁹

Chemotaxis Assay
Chemotaxis assays were performed as previously described¹⁹ with the Boyden's chamber technique. Polycarbonate filters (5-µm pore size, polyvinylpyrrolidone-free; Nucleopore Corp) were coated with 0.1% gelatin for 6 hours at room temperature. Stimuli dissolved in medium supplemented with 0.25% BSA were seeded in the lower compartment of the chamber, and 2x10⁵ suspended cells in medium containing 1% FCS were then seeded in the upper compartments. After 6 hours of incubation at 37°C, the upper surface of the filter was scraped with a rubber policeman. The filters were fixed and stained with Diff-Quick (Harleco), and 10 oil immersion fields were counted after samples were coded.

Murine Angiogenesis Assay
Female DBA2 mice (Charles River) were used at 6 to 8 weeks of age. Angiogenesis was assayed as growth of blood vessels from subcutaneous tissue into a solid gel of Matrigel containing the test sample.³⁶ Matrigel (8.1 mg protein/mL; Collaborative Research Inc) in liquid form at 4°C was mixed with the experimental substances, with or without different concentrations of heparin, and injected (0.5 mL) into the abdominal subcutaneous tissue of each mouse along the peritoneal midline. Matrigel rapidly forms a solid gel at body temperature, trapping the factors to allow slow release and prolonged exposure to surrounding tissues. At various times after this procedure mice were killed and the gels were recovered and processed for histology. Part of the tissue was fixed in 10% buffered formalin and embedded in paraffin. Sections cut at 3 µm and stained with hematoxylin and eosin were studied under light microscopy. Other sections, obtained from frozen tissue cut with a cryostat, were processed for immunofluorescence with mAb anti-L3, anti-Ly2, and anti-MAC-1 (Cederlane Laboratories Ltd), which recognize specific subsets of lymphomononuclear cells. ECs were identified by use of a polyclonal antibody anti-factor VIII–related antigen (Cederlane Laboratories Ltd) and by an mAb anti-CD31 that specifically recognizes murine ECs.³⁷ The vessel area and the total Matrigel area were planimetrically assessed from stained sections as described by Kibbey et al.³⁸ Vessels were considered to be only those structures possessing a patent lumen lined by ECs. Results for the vessel area were expressed as mean percentage±SD of the total Matrigel area.

We evaluated angiogenesis at different time intervals using as agonists HGF and its mutants and VEGF (Collaborative Research). FCE-26806 [3-(1,4,-dihydroxytetralyl)-methylene-2-oxindole; Pharmacia-Farmitalia Carlo Erba] was freshly dissolved in dimethylsulfoxide/water (6:4, vol/vol) at a concentration of 3 mmol/L and then diluted in Matrigel. The Matrigel samples were not used when FCE-26806 precipitated in the aqueous solution. A rabbit polyclonal antibody anti-VEGF (Peprotech, Inc) was diluted in Matrigel at 20 µg/mL. In preliminary experiments this dose of antibody was found to completely inhibit the angiogenesis induced by 25 ng of VEGF.

RT-PCR Analysis
Total RNA was obtained from Matrigel plugs by use of the guanidine isothiocyanate–cesium chloride method.³⁹ Two micrograms of total RNA was denatured by heating and reverse transcribed by 20 U Moloney murine leukemia virus RT into first-strand cDNA by use of 25 pmol of primers. The reaction was carried out for 1 hour at 37°C in a 20-µL final volume containing 5 mmol/L dithiothreitol, 40 U RNAsin, 5 µmol/L dNTPs mixture, and 5x buffer (200 mmol/L Tris, pH 8.3, 40 mmol/L MgCl₂). PCR was performed on a Perkin Elmer DNA thermal cycler with 5 µL of the transcription mixture and 2.5 U of Taq polymerase. dNTPs (0.2 mmol/L), 10x reaction buffer (100 mmol/L Tris-HCl, pH 8.3, 50 µmol/L KCl, 15 mmol/L MgCl₂, and 0.01% gelatin), and 35 pmol of each primer were added in a 50-µL reaction volume. The following specific oligomers (Tib Molbiol) were used:

Murine VEGF⁴⁰ : up: 5'-GGATCCATGAACTTTCTGCT-3'; down: 5'-GAATTCACCGCCTCGGCTTGTC-3'. JE⁴¹ : up: 5'-CCTGCTGCTACTCATTCA-3'; down: 5'-ATTTACGGGTCAACTTCA-3'. Murine HGF⁶ : up: 5'-TGCCCTATTTCCCGTTGT-3'; down: 5'-TTCTCCTCGCCTCTCTCA-3'. Murine MIP-2⁴² : up: 5'-GCCAGTGAACTGCGCTGTCAATGC-3'; down: 5'-GTTAGCCTTGCCTTTGTTCAGTATC-3'. KC⁴³ : up: 5'-GCCAATGAGCTGCGCTGTCAATGC-3'; down: 5'-CTTGGGGACACCTTTTAGCATCTT-3'. Murine PlGF⁴⁴ : up: 5'-CAGCAACATCACTATGCAG-3'; down: 5'-GGGTGACGGTAATAAATACG-3'. Murine met⁴⁵ : up: 5'-CCTCTCTGCCCCTTACTT-3'; down: 5'-GCTGCTGGTCTCTCGGTT-3'. Murine FGF-6⁴⁶ : up: 5'-CAGGCTCTCCTCTTCTTAG-3'; down: 5'-ATTCACACCCGAAATCTCTC-3'.

The PCR protocol for MIP-2 and KC cDNAs was as follows (numbers given are for minutes): 1 at 94°C, 1 at 55°C, and 1 at 72°C for 30 cycles, and 1 at 94°C, 1 at 55°C, and 10 at 72°C for the last cycle. For PlGF, met, HGF, and FGF-6 cDNAs the protocol was 1 at 94°C, 1 at 50°C, and 1 at 72°C for 30 cycles and 1 at 94°C, 1 at 50°C, and 10 at 72°C for the last cycle. For VEGF cDNA the protocol was 1 at 94°C, 2 at 55°C, and 3 at 72°C for 30 cycles and 1 at 94°C, 2 at 55°C, and 10 at 72°C for the last cycle. For JE cDNA the protocol was 1 at 94°C, 1 at 45°C, and 1 at 72°C for 30 cycles and 1 at 94°C, 1 at 45°C, and 10 at 72°C for the last cycle. RT-PCR of ß-actin was performed by use of specific oligonucleotides (Stratagene Cloning Systems) with the following protocol: 1 at 94°C, 1 at 55°C, and 1 at 72°C for 30 cycles and 1 at 94°C, 1 at 55°C, and 10 at 72°C for the last cycle. Twenty microliters of the amplified solution were run on a 1.8% agarose gel in Tris-borate-EDTA buffer and stained with 0.5 µg/mL ethidium bromide. PCR products were analyzed and identified by Southern blot. Specific cDNAs were labeled with [-³²P]dCTP (3000 Ci/mmol; Amersham) at 2.2x10⁸ cpm/µg specific activity by use of the random primer labeling method (Megaprime DNA labeling system, Amersham) according to the manufacturer's instructions. The gel was washed for 10 minutes in 0.5 mol/L NaOH+1.5 mol/L NaCl and for 10 minutes in 0.5 mol/L Tris (pH 7.5)+1.5 mol/L NaCl and then blotted on a nylon Duralon-UV membrane. The membrane was prehybridized for 2 hours and hybridized overnight at 42°C in a solution containing 5x Denhardt's solution, 6x SSC, 10% SDS, and 100 µg/mL denaturated salmon sperm DNA. Washes were carried out at high stringency (2x SSC+0.1% SDS at 57°C for 30 minutes, 0.5x SSC+0.1% SDS at 57°C for 30 minutes, and three times in 0.1x SSC+0.1% SDS at 57°C for 30 minutes) and the membrane was exposed to Hyperfilm MP (Amersham) with intensifying screens at -80°C for 3 days.

	Results

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In Vitro Effects of HGF and HGF Mutants on ECs
To determine the functional domains of HGF required to activate ECs in vitro, we compared the proliferating and migrating activities of HGF and three variant molecules. HGF showed a bell-shaped effect on the migration and proliferation of ECs, and the maximal biological responses were reached at 0.05 nmol/L (Figs 1 and 2). Mutants containing only the first two (HGF-NK2) or three (HGF-NK3) kringle domains of the chain did not elicit proliferation of human ECs, even when used at 8 nmol/L, a concentration 160-fold higher than that optimal for HGF (Fig 1). In contrast, both mutants elicited a weak migratory response at concentrations ranging from 4 to 8 nmol/L (Fig 2).

View larger version (14K): [in this window] [in a new window]   Figure 1. Graph shows proliferation of human ECs stimulated by different concentrations of HGF (), HGF-NK2 (), and HGF-NK3 (). Low-density cultures of endothelial cells (2.5x103 per 0.32-cm2 well) were incubated in M199 medium supplemented with 5% FCS, and fresh factor was added every 2 days. Cells were counted after 8 days. The data shown are the means (±SD) of six determinations in one representative experiment of four with similar results.

View larger version (25K): [in this window] [in a new window]   Figure 2. Graphs show migration of human ECs stimulated by different concentrations of HGF, Gln 489–HGF, HGF-NK2, and HGF-NK3. The migration of human ECs was measured by the modified Boyden's chamber technique, as described in "Methods." HGF and HGF mutants in M199 medium containing 1% FCS were seeded in the lower compartment of the chamber, and 2x105 ECs in the same medium were seeded in the upper compartment. Cells that migrated after 6 hours of incubation to the lower surface of the filter were counted after samples were coded. The numbers are the means (±SD) of three fields counted in one representative experiment of five with similar results.

HGF-NK2 and HGF-NK3 had similar effects on the murine endothelioma cell line t.End.1 (Table 1). Substitution of Arg 489 with Glu 489 at the proleolytic site blocks the cleavage of pro-HGF into the two-chain form.¹⁹ We previously reported that this mutation abolished the mitogenic effect of HGF on human ECs.¹⁹ In the present study we extended this observation to the motility of human ECs, showing that the uncleavable molecule did not induce migration. Also, the t.End.1 murine endothelioma cell line challenged with this mutant did not migrate and proliferate (Table 1).

View this table: [in this window] [in a new window]   Table 1. Effect of HGF and HGF Mutants on Proliferation and Migration of t.End.1 Cells

Angiogenic Effect in Matrigel Plug of HGF and HGF Mutants
HGF binds heparin,⁴⁷ a mucopolysaccharide known to modulate the biological response of angiogenic polypeptides.²⁹ Therefore, we first investigated the effect of heparin on HGF-induced angiogenesis. Fig 3 shows that HGF added to Matrigel induced a powerful angiogenic effect, which was enhanced by heparin. The enhancement was observed at 2 U/mL and reached the maximal effect at 10 U/mL. Higher heparin concentrations (>20 U/mL) were not used because they are angiogenic per se. On the basis of these results, we performed a series of experiments to define the angiogenic effect of HGF in the presence of 10 U/mL heparin, a concentration compatible with that reached in tissues.⁴⁸ The angiogenic activity of HGF was dose dependent, with maximal response at 36 ng/mL (0.42 nmol/L). A lower dose of HGF (3.6 ng/mL) was ineffective. When used at the optimal concentration (36 ng/mL), HGF had angiogenic activity in 85% of animals (17 positive implants of 20; P<.001 by Fisher's exact test), with maximal vascularization at day 8 (Fig 3). Time-course experiments established that at day 2 noncanalized cords of von Willebrand factor–positive ECs were predominant (Fig 4B and 4E) (23±6 von Willebrand factor–positive-ECs per microscopic field, n=8; 28±9 CD-31–positive ECs per microscopic field, n=8), and the full vascularization of the plug was reached at day 6. At day 4 canalized vessels, mainly with linear aspects, were observed (Fig 4C and 4G). These vessels were operating because they contained circulating cells. After day 6, vessels progressively assumed large and irregular aspects and at day 8 aneurismal structures with reabsorption of Matrigel were mainly present (Fig 4D). However, appearance of microaneurismal structure at day 8 did not increase the percentage of vascularized area in comparison with that observed at day 6, when these lacunae were absent. The presence of these lacunae could be due to proteolytic enzymes released by infiltrating macrophages that progressively degrade Matrigel, favoring the fusion of capillary structures. HGF-NK2 and HGF-NK3 did not cause angiogenesis when used at the optimal HGF concentration or at up to a fivefold molar excess. However, morphometric analysis showed that HGF-NK2 and HGF-NK3 had little angiogenic effect when used at 14.4 nmol/L (360 ng/mL; Fig 5). Glu 489–HGF was also unable to elicit angiogenesis at all concentrations tested (0.1 to 15 nmol/L) (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 3. Bar graphs show quantitation of angiogenesis induced by HGF. Matrigel (0.5 mL) with or without heparin was mixed with different concentrations of HGF and injected subcutaneously in mice. Quantitation was performed on hematoxylin-eosin–stained histological sections and results for the vessel area were expressed as mean percentage±SD of the total Matrigel area. A, Effect of heparin on angiogenesis induced by HGF (36 ng/mL). Animals were killed after 8 days. indicates 0 U/mL heparin; , 2 U/mL heparin; and , 10 U/mL heparin. Data are the mean±SD for four animals. *P<.05 compared with groups without heparin by paired t test. B, Dose dependence of the effect of HGF evaluated after 8 days in the presence of 10 U/mL heparin; C, Time dependence of the effect of HGF on angiogenesis in the presence of 10 U/mL heparin. Numbers in parentheses indicate the number of animals used in each group. *P<.05 by Wilcoxon's rank sum test.

View larger version (96K): [in this window] [in a new window]   Figure 4. Photomicrographs show histological analysis and immunofluorescence detection of ECs and macrophages in Matrigel plugs containing HGF (36 ng/mL) and heparin (10 U/mL). A, Control (supernatant of transfected cells with vector alone) after 8 days. B, Matrigel containing HGF after 2 days, characterized by the presence of isolated cells or clusters of cells. C, Matrigel containing HGF after 4 days with canalized vessels forming anastomotic branches. D, Matrigel containing HGF after 8 days with microaneurysmal structures containing erythrocytes. E and G, ECs stained by indirect immunofluorescence for von Willebrand factor at day 2 (E) and 4 (G). Inset of Panel E shows the section stained with secondary fluoresceinated antibody alone. Penetration of isolated and clusters of ECs (E) and ECs underlining the lumen of a vessel (G) is evident. The positivity of the cells in the human is due to the autofluorescence of the cells. F and H, Presence of MAC-1 positive cells at day 2 and day 8 detected by direct immunofluorescence. A, B, and D, original magnification x100; C, original magnification x200; E, F, and H, original magnification x250; and G, original magnification x300.

View larger version (17K): [in this window] [in a new window]   Figure 5. Bar graphs show quantitation of angiogenesis induced by HGF-NK2 (left) and HGF-NK3 (right). Matrigel (0.5 mL) containing heparin (10 U/mL) was mixed with the supernatant of N2A cells transfected with vector alone, HGF-NK2, or HGF-NK3 and injected subcutaneously in mice. Animals were killed after 8 days. Quantitation was performed on hematoxylin-eosin–stained histological sections and results for the vessel area are expressed as mean percentage±SD of the total Matrigel area. Data are the mean±SD for four animals.

In Vivo Stimulation of the HGF Receptor Kinase Is Sufficient to Induce Angiogenesis
To examine whether the HGF receptor was expressed during angiogenesis, RNA extracted by Matrigel plugs was analyzed by RT-PCR by use of specific oligonucleotides. Fig 6 shows that RNA from plugs containing HGF produced a band of 619 bp, which corresponds to the selected region of HGF receptor gene and is recognized by the specific cDNA in Southern blot analysis.

View larger version (30K): [in this window] [in a new window]   Figure 6. RT-PCR analysis shows expression of growth factors, chemokines, and met proto-oncogene in HGF-induced angiogenesis. Total RNA was extracted from the Matrigel plugs containing heparin (10 U/mL) and HGF (36 ng/mL) or heparin alone (Control) at day 4. RNA was reverse-transcripted and amplified by PCR as described in "Methods" by use of specific primers for VEGF, HGF, KC, JE, met, and ß-actin genes. Products of amplification were analyzed on a 1.8% agarose gel. The agarose gel was blotted on a nylon Duralon-UV membrane and then hybridized overnight with specific cDNAs. Three different experiments gave similar results.

The mAb DO-24 directed against the extracellular domain of the HGF receptor was used to demonstrate whether the direct stimulation of the HGF receptor was sufficient to start angiogenesis. DO-24 mAb, but not its Fab fragment, stimulated in a dose-dependent manner the in vitro proliferation and migration of human ECs (Table 2). When mixed into Matrigel, DO-24 promoted plug vascularization in a dose-dependent manner, but an mAb directed against the intracellular domain of the HGF receptor (Fig 7), the Fab–DO-24 fragment, and an irrelevant mouse IgG were ineffective (data not shown).

View this table: [in this window] [in a new window]   Table 2. Effects of DO-24 mAb on Proliferation and Migration of Human ECs

View larger version (21K): [in this window] [in a new window]   Figure 7. Bar graph shows quantitation of angiogenesis induced by DO-24 mAb. Matrigel (0.5 mL) containing heparin (10 U/mL) was mixed with different concentrations of DO-24 mAb and injected subcutaneously in mice, and angiogenesis was examined after 8 days. Quantitation was performed on hematoxylin-eosin–stained histological sections and results for the vessel area are expressed as mean percentage±SD of the total Matrigel area. Data are the mean±SD for four animals in each group. *P.05 by paired t test vs control.

To further assess the relevance of the activation of HGF receptor tyrosine kinase in angiogenesis, we conducted additional studies with the compound FCE-26806, a novel tyrosine kinase inhibitor. The inhibitor, which blocks the kinase activity of the HGF receptor,⁴⁹ was added to Matrigel with an optimal angiogenic dose of HGF or DO-24 mAb. The results obtained indicate that FCE-26806, at concentrations known to inhibit in vitro the tyrosine kinase activity of HGF,⁴⁹ inhibited the angiogenesis induced by HGF or DO-24 mAb (Table 3).

View this table: [in this window] [in a new window]   Table 3. Effect of FCE-26806 on HGF- and DO-24 mAb–Induced Angiogenesis

HGF Induces Expression of Angiogenic Factors and Chemokines
The expression in Matrigel plugs of angiogenic factors and chemokines that could enhance the angiogenesis induced by HGF has been studied by analysis of the specific transcripts. RNA extracted from plugs at day 4 was analyzed by RT-PCR by use of specific primers for PlGF, HGF, VEGF, KC, JE, and MIP-2. The transcripts for VEGF, HGF, KC, JE (Fig 6), PlGF, and MIP-2 (not shown), were present. The FGF-6 transcript was absent. The RT-PCR performed on RNA extracted from Matrigel by use of primers for VEGF gave two different products: a 580-bp and a 650-bp cDNA that correspond to murine VEGF-1 and VEGF-3.⁴⁰ To better define the role of one of these molecules present in the angiogenesis process triggered by HGF, we examined the role of VEGF by using a neutralizing antibody. The antibody anti-VEGF determined a slight but statistically significant reduction of the HGF-induced angiogenic effect (Fig 8). The relevance of the expression of chemokines, which activate functions of leukocytes,⁵⁰ was confirmed by the analysis of infiltrating cells into Matrigel plugs. Infiltrating macrophages (MAC-1–positive cells) were evident at day 2 and reached the maximum at day 4, and their number remained similar at day 8 (Fig 4F and 4H; Table 4). Few L2- and Ly3-positive lymphocytes were present (Table 4).

View larger version (18K): [in this window] [in a new window]   Figure 8. Bar graph shows effect of antibody (Ab) anti-VEGF on angiogenesis induced by HGF. Mice were injected with Matrigel containing 10 U/mL heparin supplemented with the supernatant of transfected cells with vector alone (control) or HGF (36 ng/mL). After 6 days mice were killed and plugs processed as detailed in the legend to Fig 4. Each experimental group included four mice. There was a significant difference (P<.05) between HGF and HGF+Ab anti-VEGF by Dunnet's test.

View this table: [in this window] [in a new window]   Table 4. Lymphomononuclear Infiltrate Present in Matrigel Containing HGF

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The data reported show that subnanomolar concentrations of HGF induce a marked angiogenic response in an in vivo model exploiting a reconstituted artificial basement membrane. Direct stimulation by HGF of the receptor encoded by the met proto-oncogene is sufficient to accomplish the angiogenic effect, as shown by three different experimental results: (1) the met transcript is present in the cells invading the Matrigel plug; (2) the mAb DO-24 recognizing a native epitope on the extracellular domain of the HGF receptor induces angiogenesis in vivo and proliferation and migration of ECs in vitro; and (3) FCE-26806, a molecule that blocks the tyrosine kinase activity of the HGF receptor,⁴⁹ inhibits the angiogenic effect of HGF. This compound has been demonstrated to be active in inhibiting the spontaneous vascularization of the allantochorion.⁵¹ This suggests a more general role of FCE-26806 in the control of angiogenesis other than the inhibition of biological responses elicited by the stimulation of the HGF receptor. Furthermore, the HGF action is enhanced by physiological concentrations of heparin, which can modify the interaction between HGF and the low-affinity binding sites (ie, the proteoglycans), favoring the binding to the high-affinity site, as demonstrated for FGF.²⁹ Alternatively, heparin can protect HGF from proteolytic inactivation.³⁰

To elucidate the molecular features required for EC activation and in vivo angiogenesis, we studied three different HGF mutants. A mutation that blocked the cleavage of the single-chain precursor pro-HGF into the two-chain mature form was completely defective for angiogenic activity, showing that cleavage of the precursor is required for in vivo activation of vascular cells. The recombinant proteins containing only the first two or three kringles lacked mitogenic activity in vitro, but maintained a little motogenic effect in vitro, although at a concentration 80 times higher than HGF. These data point out that HGF-induced angiogenesis requires both migration and proliferation of ECs, which are elicited only by the full-processed heterodimeric HGF molecule containing both the and ß chains. In epithelial cells, HGF-NK2 and HGF-NK3 mutants stimulate the tyrosine kinase activity of the HGF receptor but elicit only the motogenic response.^{31 52 53} An alternatively spliced form of human HGF transcript has been described^{54 55} that has a molecular structure similar to that of the HGF-NK2 mutant used in this study. The naturally occurring two-kringles spliced form inhibits HGF-induced hepatocyte proliferation in vitro.⁵⁴ Although the physiological concentration of this truncated form of HGF is not known, it is possible to speculate that this molecule may play a negative role in the control of the angiogenic process. Preliminary experiments in the Matrigel model confirm this hypothesis, because pharmacological concentrations of HGF-NK2 (4 µmol/L) partially reduce (by about 30%) the effect of HGF (F. Bussolino et al, unpublished data, 1995).

We also show that HGF induces expression of other angiogenic factors (VEGF, PlGF), including HGF itself. A neutralizing antibody anti-VEGF reduces significantly the angiogenic response to HGF, suggesting a cooperative circuit between these two molecules in the angiogenic process. Moreover, HGF-induced angiogenesis is characterized by infiltrating macrophages and expression of chemokines (JE, KC, and MIP-2). Along these lines, it has been shown that ECs stimulated with HGF express transcripts for interleukin-1 and interleukin-8⁵⁶ (the human homologs of murine KC), which stimulate monocytes and neutrophils, respectively.⁵⁰ Consequently, these data support the hypothesis that HGF elicits a complex cascade of events that may contribute to the modulation of angiogenesis into Matrigel. Therefore, one can speculate that chemokines can be released by ECs⁵⁷ or other cells not examined in this study (eg, keratinocytes or epithelial cells) and recruit macrophages. Alternatively, HGF could directly stimulate macrophages that express the MET receptor.⁵⁸ Macrophages are known to produce angiogenic mediators that indeed can amplify the action of HGF.⁵⁰

	Selected Abbreviations and Acronyms

 

ECs = endothelial cells FCS = fetal calf serum FGF = fibroblast growth factor HGF = hepatocyte growth factor mAb = monoclonal antibody MDCK = Madin-Darby canine kidney MIP-2 = macrophage inflammatory protein–2 PCR = polymerase chain reaction PlGF = placental growth factor RT = reverse transcriptase VEGF = vascular endothelial growth factor

	Acknowledgments

 
This work was supported by the CNR (Target Projects: Applicazioni Cliniche della Ricerca Oncologica and Prevention and Control of Disease Factors FATMA, subproject "Causes of infective diseases," CT 9300607.PF41); by the Istituto Superiore della Sanità (National AIDS Project, contract 9306-44); by the Associazione Italiana per la Ricerca sul Cancro; and by Telethon. Dr Silvagno and Dr Follenzi contributed equally to this work. We thank Dr W. Risau and Dr G. Breier for providing VEGF cDNA; Dr M. Persico for PlGF cDNA; Dr A. Mantovani for KC, MIP-2, and JE cDNAs and mAb MEC13 anti-murine CD-31; and Pharmacia-Farmitalia Carlo Erba for the FCE-26806 compound. We are in particular indebted to Dr W. Birchmeier, who provided the Glu 489–HGF used in the first experiments. Dr Arese is a recipient of the Fondazione Italiana per la Ricerca sul Cancro.

Received February 16, 1995; accepted July 13, 1995.

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